The immune system is composed of many interdependent cell types that collectively protect the body from bacterial, parasitic, fungal, viral infections and from the growth of tumour cells. The guards of the immune system are macrophages that continually roam the bloodstream of their host. When challenged by infection or immunisation, macrophages respond by engulfing invaders marked with foreign molecules known as antigens. This event, mediated by helper T cells, sets forth a complicated chain of responses that result in the stimulation of B-cells. These B-cells, in turn, produce proteins called antibodies, which bind to the foreign invader. The binding event between antibody and antigen marks the foreign invader for destruction via phagocytosis or activation of the complement system. A number of different classes of antibodies, also known as immunoglobulins, exist, such as IgA, IgD, IgE, IgG, and IgM. They differ not only in their physiological roles but also in their structures. From a structural point of view, IgG antibodies have been extensively studied, perhaps because of the dominant role they play in a mature immune response. Polyclonal antibodies are produced according to standard methods by immunisation of an animal with the appropriate antigen. In response, the animal will produce antibodies which are polyclonal. However, for many purposes, it is desired to have a single clone of a certain antibody, known as monoclonal antibodies. Monoclonal antibodies (MAbs) are produced by hybrid or fused cells comprised of a fusion between a normal B-cell, which produces only a single antibody, to an abnormal myeloma tumour cell. The resulting hybrid, known as a hybridoma, is these days used in standard methods for the production of antibodies.
The biological activity that the immunoglobulins possess is today exploited in a range of different applications in the human and veterinary diagnostic, health care and therapeutic sector. In fact, in the last few years, monoclonal antibodies and recombinant antibody constructs have become the largest class of proteins currently investigated in clinical trials and receiving FDA approval as therapeutics and diagnostics. Complementary to expression systems and production strategies, efficient purification protocols are required to obtain highly pure antibodies in a simple and cost-efficient manner.
Traditional methods for isolation of immunoglobulins are based on selective reversible precipitation of the protein fraction comprising the immunoglobulins while leaving other groups of proteins in solution. Typical precipitation agents are ethanol, polyethylene glycol, lyotropic salts such as ammonium sulphate and potassium phosphate, and caprylic acid. Typically, these precipitation methods are giving very impure products while at the same time being time consuming and laborious. Furthermore, the addition of the precipitating agent to the raw material makes it difficult to use the supernatant for other purposes and creates a disposal problem, which is particularly relevant when speaking of large-scale purification of immunoglobulins.
An alternative method for isolation of immunoglobulins is chromatography, which embraces a family of closely related separation methods. The feature distinguishing chromatography from most other physical and chemical methods of separation is that two mutually immiscible phases are brought into contact wherein one phase is stationary and the other mobile. The sample mixture, introduced into the mobile phase, undergoes a series of interactions with the stationary and mobile phases as it is being carried through the system by the mobile phase. Interactions exploit differences in the physical or chemical properties of the components in the sample. These differences govern the rate of migration of the individual components under the influence of a mobile phase moving through a column containing the stationary phase. Separated components emerge in the order of increasing interaction with the stationary phase. The least retarded component elutes first, the most strongly retained material elutes last. Separation is obtained when one component is retarded sufficiently to prevent overlap with the zone of an adjacent solute as sample components elute from the column. Efforts are continuously being made to design the optimal stationary phase for each specific separation purpose. Such a stationary phase is commonly comprised of a support or base matrix to which a ligand comprising functional i.e. binding groups has been attached. Reference is commonly made to each kind of chromatography based on the principle of interaction it utilises, such as ion-exchange chromatography, hydrophobic interaction chromatography and affinity chromatography.
Ion exchange chromatography is frequently used in protocols for the isolation of immunoglobulins. In anion exchange chromatography, negatively charged amino acid side chains of the immunoglobulin will interact with positively charged ligands of a chromatography matrix. In cation exchange chromatography on the other hand, positively charged amino acid side chains of the immunoglobulin will interact with negatively charged ligands of a chromatography matrix.
Hydrophobic interaction chromatography (HIC) is another method described and used in protocols for the isolation of immunoglobulins. If a highly pure immunoglobulin product is the object, it is commonly recommended to combine HIC with one or more further steps. In HIC, in order to make the immunoglobulin bind efficiently to the HIC matrix, addition of lyotropic salts to the mobile phase is required. The bound immunoglobulin is subsequently released from the matrix by lowering the concentration of lyotropic salt. Thus, a disadvantage of this procedure is the necessity to add lyotropic salt to the raw material, as this may cause problems and a consequently increased cost to the large-scale user. For example, for raw materials such as whey, plasma, and egg yolk, the addition of lyotropic salts to the raw materials would in many instances be prohibitive in large-scale applications, as the salt could prevent any economically feasible use of the immunoglobulin depleted raw material. An additional problem in large-scale applications would be the disposal of several thousand litres of waste.
Affinity chromatography is based on specific interactions between a target biomolecule and a biospecific ligand in a principle of lock-key recognition. Thus, the target and ligand will constitute an affinity pair, such as antigen/antibody, enzyme/receptor etc. Protein-based affinity ligands are well known, such as Protein A and Protein G affinity chromatography which are both widespread methods for isolation and purification of antibodies. It is well known that Protein A chromatography provides an outstanding specificity, particularly towards monoclonal antibodies, and consequently high purities are obtainable. Used in combination with ion exchange, hydrophobic interaction, hydroxyapatite and/or gel filtration steps, Protein A-based methods have become the antibody purification method of choice for many biopharmaceutical companies, see e.g. WO 8400773 and U.S. Pat. No. 5,151,350. However, due to the peptide bonds of the proteins, protein A matrices present a certain degree of alkaline sensitivity. In addition, when Protein A matrices are used to purify antibodies from cell culture media, proteases originating from the cells may cause leakage of Protein A, or peptide fragments thereof.
An attempt to reduce ligand leakage from affinity chromatography matrices has been is presented in WO 03/041859 (Boehlinger Ingelheim Pharma KG), wherein it is suggested to pretreat e.g. Protein A matrices with at least one surfactant to reduce ligand leakage. The affinity matrix may be treated e.g. with 5-15 bed volumes of surfactant. The contact time is crucial for the effectiveness of the process. For example, at room temperature, a contact time of at least 16 h is required for a reduction in leakage.
An alternative approach to the problem of ligand leakage from affinity chromatography matrices is provided in U.S. Pat. No. 4,983,722 (Miles Inc.), wherein Protein A is selectively isolated from a liquid containing antibody and Protein A by exposure thereof to an anion exchange material. Both components are adsorbed to the anion-exchange material, and the antibodies and Protein A are then sequentially eluted under conditions of increasing ionic strength. An illustrative anion exchanger is diethylaminoethyl (DEAE) Trisacryl M or DEAE Sepharose™.
WO 2004/076485 (Lonza Biologics Plc.) relates to antibody purification by Protein A and ion exchange chromatography. The ion exchange step comprises loading the antibody purified on Protein A on an ion exchange material under conditions which allow for the binding of Protein A and collecting the antibody in the flow-through. The anion exchanger is a quaternary amine-based anion exchanger, most preferably Sepharose™ Q (Amersham Biosciences, now GE Healthcare).
U.S. Pat. No. 5,429,746 (SmithKline Beecham Corp.) relates to a process wherein the antibody is first adsorbed to a Protein A chromatographic support and eluted; then adsorbed to a cation exchange chromatographic support and selectively eluted there from; and finally adsorbed to a HIC support and eluted. The mixture applied to the HIC column, following affinity and/or cation exchange chromatography, may contain immunoglobulin aggregates, misfolded species, host cell proteins and residue material from the affinity chromatography step.
U.S. Pat. No. 6,498,236 (Upfront Chromatography) is directed to specific problems caused by small differences in molecular weight between protein-based affinity ligands and target immunoglobulins. Thus, a method is disclosed for the isolation or purification of immunoglobulins from a solution, such as a hybridoma cell culture supernatant, animal plasma or ser, which method is suggested as an alternative to the use of Protein A, Protein G, synthetic peptides and other relatively high molecular weight ligands. The solid phase matrices used in the disclosed method are defined by the formula M-SP1-X-A-SP2-ACID, wherein M designates the matrix backbone, SP1 designates a spacer, X designates O, S or NH, A designates a mono- or bicyclic optionally substituted aromatic or heteroaromatic moiety, SP2 designates an optional spacer and ACID designates an acidic group. The specific substituents are stated to be decisive as to whether the matrix will be binding immunoglobulins efficiently.
U.S. Pat. No. 5,945,520 (Burton et al) discloses mixed mode chromatographic resins which exhibit a hydrophobic character at the pH of binding and a hydrophilic and/or electrostatic character at the pH of desorption. The resin is specifically designed to bind the target compound from an aqueous solution at both a low and high ionic strength. Thus, the adsorption step utilises HIC, while desorption is based on charge repulsion.
U.S. Pat. No. 6,702,943 (Johansson et al) discloses a method for removal of a target substance from a liquid by adsorption thereof to a matrix carrying a plurality of ligands comprising anion-exchanging groups and a hydrophobic structure. More specifically, the ligands contain an aromatic ring in the proximity of the positively charged anion-exchanging groups. It is stated that inclusion of other groups capable of electron donor-electron acceptor interactions may enhance the strength of the interaction between the substance and the adsorbent. The desired substances are stated to be cells, parts of cells and substances comprising peptide structures. The break-through capacity of the matrix is defined for reference proteins such as bovine serum albumin and IgG. The ligands disclosed are denoted “high salt ligands” due to their capability of adsorbing target substances at high concentrations of salt such as 0.25M NaCl.
Further, WO 01/38228 (Belew et al.) discloses another method for removal of a negatively charged substance from a liquid by binding thereof to a matrix that comprises mixed mode anion-exchanging ligands. Each ligand comprises a positively charged nitrogen and a thioether linkage at a distance of 1-7 atoms from said charged nitrogen. Similar to the above, the desired substances, such as cells, parts of cells and substances comprising peptide structures are adsorbed at salt concentrations in the region of 0.25M NaCl.
Ceramic hydroxyapatite has been suggested as useful for immunoglobulin polishing. More specifically, it has been reported (Chromatography, tech note 2849; S. G. Franklin, Bio-Rad Laboratories, Inc., 2000 Alfred Nobel Drive, Hercules, Calif. 94547 USA) that IgG1 can be resolved from an IgG1-Protein A complex in unfractionated media on CHT ceramic hydroxyapatite (Bio-Rad). More specifically, hydroxyapatite (Ca10(PO4)6(OH)2) is a form of calcium phosphate, which has been shown to possess unique separation properties. However, hydroxyapatite-based matrices are also known to involve certain disadvantages. For example, due to Ca-leakage, they are unstable at acidic pH values, and they are sensitive to chelating agents such as EDTA. In addition, it has been shown that it is difficult to develop, and to scale up, a robust and reproducible purification method using hydroxyapatite-based matrices, e.g. because it has been difficult to pack hydroxyapatite, and to maintain the performance in large columns. Finally, there is a risk of alterations of the resin properties caused by metal ion contamination and exchange of calcium ions, which alteration is a serious concern for regulatory authorities.
Johansson et al (Journal of Chromatography A, 1016 (2003) 21-33: “Preparation and characterization of prototypes for multi-modal separation media aimed for capture of negatively charged biomolecules at high salt conditions”) describes screening of prototypes of multi-modal ligands for the capture of negatively charged proteins from high conductivity mobile phases. It was found that non-aromatic multi-modal anion-exchange ligands based on weak ion-exchange ligands (primary and secondary amines) were optimal for the capture of proteins by adsorption at high salt conditions.